The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare.
The present invention relates to the production and isolation of novel cellulases exhibiting optimum activity of the C.sub.1 and C.sub.x types at temperatures in the range of about 60.degree. to about 70.degree. C and under moderately acid conditions, by the cultivation of the fungus Thielavia terrestris in a suitable cellulose-containing medium.
The term cellulase as employed hereinafter designates an enzyme complex capable of depolymerizing and hydrolyzing cellulose to glucose. Those cellulases known to the prior art have been produced either by fungi or by certain mold-like higher bacteria.
Various of these types of organisms have been shown to possess the ability to grow on a cellulose substrate. Many fungal cellulases are now commercially available. They are produced mostly by mesophilic fungi, i.e., those which will grow well at intermediate temperatures (25.degree. to 37.degree. C), and some of these cellulases exhibit relatively high C.sub.1 -enzyme type activity, meaning that they are capable of depolymerizing natural cellulose. The disadvantage of these fungal cellulases is that they cannot ordinarily be used to degrade cellulose at temperatures higher than about 50.degree. C owing to their lack of heat stability.
Another type of cellulase activity is C.sub.x -enzyme activity, measured by an ability to decompose carboxymethylcellulose (CMC). C.sub.1 type activity is of primary commercial significance.
Cellulases produced by various organisms have been described in U.S. Pat. Nos. 3,232,832 (Rhizopus); 3,398,055 (Trichoderma viride ATCC 16325, and other fungi); 3,438,864 (Eumyces mold organism ATCC 16425); 3,677,899 (Lampteromyces or Formitopsis); and 3,734,831. However, cellulase preparations from Trichoderma viride, while known to be higher in C.sub.1 -enzyme activity than any other commercially available cellulases, have the drawback that the optimum temperature for their enzymic reactions is about 50.degree. C (Mandels, et al. "Cellulases and Their Applications", ACS, Vol. 95, pp. 398-411 (1969)).
U.S. Pat. No. 3,812,013 discloses production of a soluble cellulase which is said to display optimum activity at pH levels from 5.5 to 8 at temperatures of 65.degree. to 80.degree. C, from a thermophilic actinomyces which is not otherwise specifically identified, and under conditions requiring the introduction of an oxygenating gas into the culture medium so as to maintain a minimum dissolved oxygen level therein. However, actinomyces are not true fungi, but are mold-like branching filamentary higher bacteria (Burdon and Williams, "Microbiology", 5th edition, 1964).
A publication by D. Knoesel and A. Resz, "Fungi from Compost of Waste Material", in Staedthygiene, 1973, 24(6), pp. 143-8; Chem. Abst. 79: 123441h (1973), discloses strains of numerous genera of thermophilic and thermotolerant fungi capable of decomposing natural cellulose, and suggests the presence of enzymes with C.sub.1 and C.sub.x activities, including: Mucor, Absidia, Talaromyces, Dactylomyces, Myriococcum, Humicola, Thermoidium, Aspergillus, Paecilomyces, Scopulariopsis, and Acremonium.
F. J. Stutzenberger, Appl. Microbiol. 1971, 22 (2), pp. 147-152; Chem. Abst. 75: 106381p (1971), mentions cellulase production by a cellulytic thermophilic actinomycete, Thermonospora curvata, at optimum of pH 6.0 and temperature 55.degree. to 65.degree. C.
B. Flannigan and P. N. Sellars, Trans. Brit. Mycol. Soc. 1972, 58 (pt. 2), pp. 338-341; Chem. Abst. 77: 45327t (1972) discloses the use of the thermophilic fungus Thielavia sepedonium P101 to hydrolyze carboxymethylcellulose, but does not describe the production of a cellulase(s) therefrom, or its (their) properties.